Patient care as well as the prevention and transmission of Hepatitis C Virus (HCV) by blood and blood products or by close personal contact require sensitive, specific methods for screening and identifying carriers of HCV and HCV-contaminated blood or blood products. Serological determination of HCV exposure relies upon the detection of anti-HCV antibodies present in human blood plasma or sera. These anti-HCV antibodies are directed against a number of distinct structural and non-structural proteins encoded by the virus.
The genomic sequence of HCV is known, as are methods for obtaining the sequence. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviviridae family of viruses. Based on phylogenetic analysis, at least six distinct, but related, genotypes of HCV have been identified (Simmonds et al., J. Gen. Virol. 74: 2391-2399 (1993)).
The virus encodes a single polyprotein having more than 3,000 amino acid residues (Choo et al., Science 244: 359-362 (1989)). The polyprotein is processed co- and post-translationally into both structural and non-structural proteins. The polyprotein is cleaved into the following products: NH2-C-E1-E2-P7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial cleavage of the polyprotein is catalyzed by host proteases, which liberate three structural proteins, the N-terminal nucleocapsid protein (called “core” and designated “C”) and two envelope glycoproteins “E1” (also known as E) and “E2” (also known as E2/NS1), as well as nonstructural (NS) proteins that contain the viral enzymes. The NS regions are termed NS2, NS3, NS4, NS4a, NS4b, NS5a and NS5b.
Most commercial serological assays utilize an indirect format in which anti-HCV antibodies are captured by recombinant HCV antigens present on a solid phase, followed by detection of the anti-HCV antibody by a labeled anti-human antibody conjugate. While some of the antigenic regions of HCV have been identified, peptides and recombinant proteins from these regions exhibit a variable degree of sensitivity and selectivity in detection and diagnosis of HCV carriers. HC43 is one such recombinant protein used for the detection of HCV antibodies in human serum or plasma (SEQ ID NO: 4). HC43 contains the 33c region of the NS3 protein (HCV-1 amino acids 1192-1457) and the core or nucleocapsid structural protein (HCV-1 amino acids 1-150). HC43 is expressed in E. coli as a non-fusion protein by using a plasmid (pKRR826) containing the pL promoter of bacteriophage lambda (described in U.S. Pat. No. 6,846,905), utilizing a codon-optimized sequence from the HCV H strain (i.e., HCV-1; Ogata et al., PNAS USA 88: 3392-3396 (1991)). Two non-HCV coding amino acids separate the NS3 and core sequences. Another such recombinant protein used for the detection of anti-HCV antibodies is C100. This recombinant protein is derived from the NS4 region of the HCV genome (HCV amino acids 1569-1931), and is expressed in yeast with an N-terminal superoxide dismutase (SOD) fusion of 527 amino acids (see, e.g., U.S. Pat. No. 5,350,671). Although 363 amino acids of the HCV genome are present in the recombinant protein, studies have demonstrated that the majority of antibody binding occurs in two smaller regions within the NS4 region. The first region is the 5-1-1 region, which comprises HCV amino acids 1691-1733, and the second is the C100 region compromising HCV amino acids 1921-1940.
Previous attempts to express large regions of the HCV nonstructural regions (i.e., amino acids 1569-1931), including NS4a and NS4b, resulted in poor expression in E. coli. Thus, there is a need for HCV antigens, which comprise core and NS4 epitopes immunoreactive with anti-HCV antibodies and which can be expressed at high levels in bacteria, such as E. coli. 
In view of the foregoing, the present disclosure seeks to provide such HCV antigens. This and other objects and advantages, as well as additional inventive features, will become apparent from the detailed description provided herein.